Modified Saponins for the Treatment of Fungal Infections

ABSTRACT

Methods of treating a fungal infection in a subject, the method comprising administering to the subject a modified saponin.

CLAIM OF PRIORITY

This application claims the benefit of U.S. Provisional PatentApplication Ser. No. 61/294,304, filed on Jan. 12, 2010, the entirecontents of which are hereby incorporated by reference.

FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

This invention was made with Government support under Grant No. R01AI075286 awarded by the National Institutes of Health, and the NationalCancer Institute's Initiative for Chemical Genetics under contract#N01-CO-12400. The Government has certain rights in the invention.

TECHNICAL FIELD

This invention relates to methods of treating fungal infections usingsaponins disclosed herein.

BACKGROUND

Fungal infections are a major cause of morbidity and mortality and thereis an urgent need for the development of new antifungal agents.Candidiasis is the most common fungal infection and Candida spp. havebecome the fourth leading cause of bloodstream infections in the UnitedStates (Edmond et al., Clin Infect Dis, 29:239-244 (1999); Pfaller etal., Clin Microbiol Rev, 20:133-163 (2007)). In addition to themorbidity and mortality associated with systemic candidiasis, localizedinfections are also a significant health issue. Candida spp. arc thesecond most common cause of urinary tract infection (Laupland et al., JCrit Care, 17:50-57 (2002)) and according to different studies,approximately 70% of women experience vaginal infections caused byCandida spp., 20% of them suffer from recurrent infections, and of theselatter recurrent infections, about half of the patients have four ormore episodes per year (Paulitsch et al., Mycoses, 49:471-475 (2006);Corsello et al., Eur J Obstet Gynecol Reprod Biol, 110:66-72 (2003);Ventolini et al., J Reprod Med, 51:475-478 (2006)).

The success of Candida albicans as a human pathogen is a result of theirdiverse armamentarium of virulence factors. C. albicans colonizesmucosal surfaces, such as the gastrointestinal tract (isolated from overhalf of the oral cavities of healthy adults) and vaginal epithelium(Paulitsch et al., Mycoses, 49:471-475 (2006); Kumamoto et al., Annu RevMicrobiol, 59:113-133 (2005); Li et al., Microbiology, 149:353-362(2003)). Candida virulence is a result of its ability to form biofilms,switch between different forms, and produce filaments in response toenvironmental conditions (Berman et al., Nat Rev Genet, 3:918-932(2002); Kobayashi et al., Trends Microbiol, 6:92-94 (1998)). Candidabiofilm formation has important clinical repercussions because of theirincreased resistance to antifungal therapy and the ability of cellswithin biofilms to withstand host immune defenses, resulting intreatment failure and the need to remove catheters and other biologicalmaterials (Kumamoto et al., Annu Rev Microbiol, 59:113-133 (2005); Kojicet al., Clin Microbiol Rev, 17:255-267 (2004); Raad, Middle East JAnesthesiol, 12:381-403 (1994); Ramage et al., FEMS Yeast Res, 6:979-986(2006); Richard et al., Eukaryot Cell, 4:1493-1502 (2005)).

SUMMARY

The present invention is based, at least in part, on the discovery ofsaponins that are active antifungals; without wishing to be bound bytheory, it is believed that these saponins exert their activity eitherdirectly and/or by enhancing the host antifungal responses. Thus,described herein are compounds for use in the treatment of fungalinfections, e.g., Candida infections. Also described are methods fortreating fungal infections, e.g., Candida infections, by administering atherapeutically effective amount of a saponin as described herein.

In one aspect, provided herein are compounds selected from the groupconsisting of aginosides, arvensoside B, barrigenols, sakurasosaponinsand maesabalides for use in the treatment of fungal infections. Alsoprovided are methods of treating a fungal infection in a subject. Themethods include administering to the subject a therapeutically effectiveamount of a saponin, e.g., a compound selected from the group consistingof aginosides, arvensoside B, barrigenols, sakurasosaponins andmaesabalides.

In another aspect, the present invention features methods for treatingfungal infections in a subject, by administering a compound of FormulaI:

or a pharmaceutically acceptable salt thereof, wherein:

R¹ and R² are each independently H or OH;

Gly is

R³ is OH or

R⁴ and R⁵ are each independently OH or

and

denotes either a single bond or a double-bond.

In some embodiments, the compound is:

or a pharmaceutically acceptable salt thereof.

In some embodiments, the compound is:

or a pharmaceutically acceptable salt thereof.

In some embodiments, the compound is:

-   -   or a pharmaceutically acceptable salt thereof.

In some embodiments, the compound is:

or a pharmaceutically acceptable salt thereof.

In some embodiments, the compound is:

or a pharmaceutically acceptable salt thereof.

In another aspect, the invention provides methods for treating a fungalinfection in a subject. The methods include administering a compound ofFormula II:

or a pharmaceutically acceptable salt thereof, wherein:

R₁ is H or OC(O)R^(A);

R₂ is H or OH;

R₃ is H, C(O)OR^(A), CH₂OR^(A), CH₂OC(O)R^(A);

R₄ and R₅ are each independently H or OC(O)R^(B);

R^(A) is H or C₁₋₆ alkyl;

R^(B) is C₂₋₆ alkenyl; and

Gly is

In some embodiments, the compound is:

or a pharmaceutically acceptable salt thereof.

In some embodiments, the compound is:

or a pharmaceutically acceptable salt thereof.

In some embodiments, the compound is:

or a pharmaceutically acceptable salt thereof.

In some embodiments, the compound is:

or a pharmaceutically acceptable salt thereof.

In yet a further aspect, the invention provides methods for treating afungal infection in a subject. The methods include administering to thesubject a compound of Formula III:

or a pharmaceutically acceptable salt thereof, wherein:

R¹ is H or C(O)R^(B);

R² is C₁₋₆ alkyl or aryl; and

R^(B) is C₂₋₆ alkenyl.

In some embodiments, the compound is:

or a pharmaceutically acceptable salt thereof.

In some embodiments, the compound is:

or a pharmaceutically acceptable salt thereof.

In yet another aspect, the invention provides methods for treating afungal infection in a subject. The methods include administering to thesubject a compound:

or a pharmaceutically acceptable salt thereof.

Also provided herein is the saponins described herein for use in thetreatment of a fungal infection, and/or in the manufacture of amedicament for the treatment of a fungal infection.

In some embodiments of the methods described herein, the fungalinfection is infection with a Candida species fungus, e.g., C. albicans.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Methods and materials aredescribed herein for use in the present invention; other, suitablemethods and materials known in the art can also be used. The materials,methods, and examples are illustrative only and not intended to belimiting. All publications, patent applications, patents, sequences,database entries, and other references mentioned herein are incorporatedby reference in their entirety. In case of conflict, the presentspecification, including definitions, will control.

Other features and advantages of the invention will be apparent from thefollowing detailed description and figures, and from the claims.

DESCRIPTION OF DRAWINGS

FIGS. 1A-C show structures of the natural product saponins identified inthe C. elegans-C. albicans antifungal drug discovery screen (allstructural representations were provided by Analyticon Discovery,Germany). For each of the compounds the maximum nematode survival (%) isindicated.

FIGS. 2A-B are line graphs showing the dose response of select compoundsidentified in the C. elegans-C. albicans assay. 2A, Two saponins (A7 andA20) were as effective as amphotericin B in promoting C. eleganssurvival. The decrease in nematode survival for A7 at the highestconcentration tested suggests the saponin maybe toxic to the nematode2B, Dose response of two saponin compounds (A16 and A19) used in furtherstudies.

FIG. 3 is a bar graph showing biofilm formation for two saponin familymembers identified in the screen compared to untreated silicone pads andcaspofungin, a compound able to inhibit C. albicans biofilm formation.Standard deviations are depicted and based on 5-11 silicone padmeasurements.

FIGS. 4A-C are line graphs showing phototoxicity in C. albicans DAY185after incubation with or without 4 μg/ml A16 and (4A) 100 μM RB, (4B)100 μM ce6, and (4C) 10 μM PEI-ce6. Fungal cells were incubated with thePS for 30 min, washed and then illuminated and survival fractions weredetermined as described in the methods. Values are means of threeseparate experiments and bars are SEM. *** P<0.001 compared to PS alone.

FIGS. 5A-D are confocal laser scanning microscope images of C. albicanscells after incubation with (5A) 100 μM ce6 and in combination with 4μg/ml A16 for (5B) 1 hr and (5C) (5D) 24 hrs. Scale bar=20 μm for (5A),(5B), and (5C) and 14 μm for (5D).

FIGS. 6A-B are the structures of two clinically relevant antifungalagents. 6A, The polyene antifungal amphotericin B; 6B, caspofungin, amember of the echinocandin antifungal family.

DETAILED DESCRIPTION

A compound screen to identify potential antifungal natural products wasundertaken, identifying 12 saponins, some of which have not beenpreviously described. This class of amphipathic natural products wasrepresented by members of the maesabalide and barrigenol families, aswell as others. In the Caenorhabditis elegans model, some saponinsconferred nematode survival comparable to amphotericin B. Of the 12antifungal saponins identified, two were selected for further analysis.C. albicans isolates were inhibited by these compounds at relatively lowconcentrations (16 and 32 μg/mL) including isolates resistant toclinically used antifungal agents. C. albicans hyphae and biofilmformation were also disrupted in the presence of these natural products,and studies demonstrate that fungal cells in the presence of saponinsare more susceptible to salt induced osmotic stress. Although saponinsare known for their hemolytic activity, we observed no hemolysis oferythrocytes at three times the minimal inhibitory concentration (100μg/mL) for C. albicans, suggesting the saponins may have a preferencefor binding to fungal ergosterol when compared to cholesterol.Importantly, when used in combination with photosensitizer compounds,the fungus displayed increased susceptibility to photodynamicinactivation due to the ability of the saponins to increase cellpermeability facilitating penetration of the photosensitizers. The largeproportion of compounds identified as antifungal agents containingsaponin structural features suggests it may be a suitable chemicalscaffold for a new generation of antifungal compounds.

There is an urgent need for the development of new antifungal agents[reviewed in Spanakis et al., Clin Infect Dis, 43:1060-1068 (2006)].Traditionally, natural products have provided a plethora ofantimicrobial compounds. In particular, a current drug of choice fortreatment of systemic candidiasis is the polyene amphotericin B (FIG. 6,panel a) originally isolated from Streptomyces nodosus Trejo (Gold etal., Antibiotics Ann, 1955-1956, 579-586; Trejo et al., J Bacteriol,85:436-439 (1963)). Plants are also well known to produce a diversearray of natural products which harbor antimicrobial activity (Dixon,Nature, 411:843-847 (2001)), including phytoalexins and saponins.

Saponins

Saponins have been identified in over one hundred plant families and canbe an integral part of the plant's defense mechanism. These naturalproducts are composed of sugar moieties connected to a hydrophobicaglycone backbone. Various side chains to both the aglycone and thependant sugar moieties create additional structural diversity. Saponinsare able to form pores in lipid bilayers and are known to increasecellular permeability allowing uptake of molecules that would otherwisebe excluded. In this report we utilized the nematode Caenorhabditiselegans as a heterologous host to screen a library of natural products(Breger et al., PLoS Pathog, 3:e18 (2007); Okoli et al., PLoS ONE,4:e7025 (2009)), ultimately identifying twelve saponins which increasednematode survival. Some saponins were able to prolong nematode survivalin a dose-dependent manner and further characterization of theantifungal activity of members of the saponin family demonstrate theycan impede C. albicans biofilm formation and dramatically potentiatephotodynamic inactivation (PDI) when coupled with photosensitizers (PSs)and harmless visible light. These compounds may be antifungal agents forclinical use either by themselves, or in conjunction with currently usedantifungal agents.

Methods of Treatment

The methods described herein include methods for the treatment ofdisorders associated with fungal infections, e.g., infections withCandida albicans. Generally, the methods include administering atherapeutically effective amount of a therapeutic saponin compound asdescribed herein, to a subject who is in need of, or who has beendetermined to be in need of, such treatment.

As used in this context, to “treat” means to ameliorate at least onesymptom of the disorder associated with a fungal infections. Often, afungal infections results in redness, itching. Discharge, and/ordiscomfort; thus, a treatment can result in a reduction in a reductionin redness, itching, discharge, and/or discomfort. Administration of atherapeutically effective amount of a compound described herein for thetreatment of a condition associated with fungal infections will resultin decreased levels of fungal organism present, and a reduction insymptoms if present.

Candidiasis

In some embodiments, the disorder is Candidiasis, e.g., oral thrush,vaginitis, or systemic candidiasis, e.g., candidemia. Most candidainfections are minor and result in minimal complications such asredness, itching and discomfort, though the infections can be severe oreven fatal if left untreated in certain populations, such as inimmunocompetent persons. Candidiasis is usually a localized infection,e.g., of the skin or mucosal membranes, e.g., the oral cavity, thepharynx or esophagus, the gastrointestinal tract, the urinary bladder,or the genitalia. Walsh and Dixon, “Deep Mycoses,” in Baron et al. eds.Baron's Medical Microbiology (4th ed.). Univ of Texas Medical Branch(1996).

In immunocompromised patients, Candida infections can affect theesophagus with the potential of becoming systemic, causing the much moreserious fungemia called candidemia Immunocompromised patients includethose with metabolic illnesses such as diabetes, or with weakened orundeveloped immune systems; diseases or conditions linked to candidiasisinclude HTV/AIDS, mononucleosis, cancer treatments, steroids, stress,and nutrient deficiency.

Diagnosis of Candida infections can be done using methods known in theart, e.g., via microscopic examination or culturing a sample suspectedof containing the infections organism.

Dosage

An “effective amount” is an amount sufficient to effect beneficial ordesired results. For example, a therapeutic amount is one that achievesthe desired therapeutic effect. This amount can be the same or differentfrom a prophylactically effective amount, which is an amount necessaryto prevent onset of disease or disease symptoms. An effective amount canbe administered in one or more administrations, applications or dosages.A therapeutically effective amount of a therapeutic compound (i.e., aneffective dosage) depends on the therapeutic compounds selected. Thecompositions can be administered one from one or more times per day toone or more times per week; including once every other day. The skilledartisan will appreciate that certain factors may influence the dosageand timing required to effectively treat a subject, including but notlimited to the severity of the disease or disorder, previous treatments,the general health and/or age of the subject, and other diseasespresent. Moreover, treatment of a subject with a therapeuticallyeffective amount of the therapeutic compounds described herein caninclude a single treatment or a series of treatments.

Dosage, toxicity and therapeutic efficacy of the therapeutic compoundscan be determined by standard pharmaceutical procedures in cell culturesor experimental animals, e.g., for determining the LD50 (the dose lethalto 50% of the population) and the ED50 (the dose therapeuticallyeffective in 50% of the population). The dose ratio between toxic andtherapeutic effects is the therapeutic index and it can be expressed asthe ratio LD50/ED50. Compounds which exhibit high therapeutic indicesare preferred. While compounds that exhibit toxic side effects may beused, care should be taken to design a delivery system that targets suchcompounds to the site of affected tissue in order to minimize potentialdamage to uninfected cells and, thereby, reduce side effects.

The data obtained from cell culture assays and animal studies can beused in formulating a range of dosage for use in humans. The dosage ofsuch compounds lies preferably within a range of circulatingconcentrations that include the ED50 with little or no toxicity. Thedosage may vary within this range depending upon the dosage formemployed and the route of administration utilized. For any compound usedin the method of the invention, the therapeutically effective dose canbe estimated initially from cell culture assays. A dose may beformulated in animal models to achieve a circulating plasmaconcentration range that includes the 1050 (i.e., the concentration ofthe test compound which achieves a half-maximal inhibition of symptoms)as determined in cell culture. Such information can be used to moreaccurately determine useful doses in humans. Levels in plasma may bemeasured, for example, by high performance liquid chromatography.

Pharmaceutical Compositions and Methods of Administration

The methods described herein include the manufacture and use ofpharmaceutical compositions, which include saponins described herein asactive ingredients. Also included are the pharmaceutical compositionsthemselves.

Pharmaceutical compositions typically include a pharmaceuticallyacceptable carrier. As used herein the language “pharmaceuticallyacceptable carrier” includes saline, solvents, dispersion media,coatings, antibacterial and antifungal agents, isotonic and absorptiondelaying agents, and the like, compatible with pharmaceuticaladministration. Supplementary active compounds can also be incorporatedinto the compositions, e.g., other anti-fungal agents.

Pharmaceutical compositions are typically formulated to be compatiblewith its intended route of administration. Examples of routes ofadministration include parenteral, e.g., intravenous, intradermal,subcutaneous, oral (e.g., inhalation), transdermal (topical),transmucosal, vaginal and rectal administration.

Methods of formulating suitable pharmaceutical compositions are known inthe art, see, e.g., the books in the series Drugs and the PharmaceuticalSciences: a Series of Textbooks and Monographs (Dekker, N.Y.). Forexample, solutions or suspensions used for parenteral, intradermal,vaginal or subcutaneous application can include the followingcomponents: a sterile diluent such as water for injection, salinesolution, fixed oils, polyethylene glycols, glycerine, propylene glycolor other synthetic solvents; antibacterial agents such as benzyl alcoholor methyl parabens; antioxidants such as ascorbic acid or sodiumbisulfite; chelating agents such as ethylenediaminetetraacetic acid;buffers such as acetates, citrates or phosphates and agents for theadjustment of tonicity such as sodium chloride or dextrose. pH can beadjusted with acids or bases, such as hydrochloric acid or sodiumhydroxide. The parenteral preparation can be enclosed in ampoules,disposable syringes or multiple dose vials made of glass or plastic.

Pharmaceutical compositions suitable for injectable use can includesterile aqueous solutions (where water soluble) or dispersions andsterile powders for the extemporaneous preparation of sterile injectablesolutions or dispersion. For intravenous administration, suitablecarriers include physiological saline, bacteriostatic water, CremophorEL™ (BASF, Parsippany, N.J.) or phosphate buffered saline (PBS). In allcases, the composition must be sterile and should be fluid to the extentthat easy syringability exists. It should be stable under the conditionsof manufacture and storage and must be preserved against thecontaminating action of microorganisms such as bacteria and fungi. Thecarrier can be a solvent or dispersion medium containing, for example,water, ethanol, polyol (for example, glycerol, propylene glycol, andliquid polyetheylene glycol, and the like), and suitable mixturesthereof. The proper fluidity can be maintained, for example, by the useof a coating such as lecithin, by the maintenance of the requiredparticle size in the case of dispersion and by the use of surfactants.Prevention of the action of microorganisms can be achieved by variousantibacterial and antifungal agents, for example, parabens,chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. In manycases, it will be preferable to include isotonic agents, for example,sugars, polyalcohols such as mannitol, sorbitol, sodium chloride in thecomposition. Prolonged absorption of the injectable compositions can bebrought about by including in the composition an agent that delaysabsorption, for example, aluminum monostearate and gelatin.

Sterile injectable solutions can be prepared by incorporating the activecompound in the required amount in an appropriate solvent with one or acombination of ingredients enumerated above, as required, followed byfiltered sterilization. Generally, dispersions are prepared byincorporating the active compound into a sterile vehicle, which containsa basic dispersion medium and the required other ingredients from thoseenumerated above. In the case of sterile powders for the preparation ofsterile injectable solutions, the preferred methods of preparation arevacuum drying and freeze-drying, which yield a powder of the activeingredient plus any additional desired ingredient from a previouslysterile-filtered solution thereof.

Oral compositions generally include an inert diluent or an ediblecarrier. For the purpose of oral therapeutic administration, the activecompound can be incorporated with excipients and used in the form oftablets, troches, or capsules, e.g., gelatin capsules. Oral compositionscan also be prepared using a fluid carrier for use as a mouthwash.Pharmaceutically compatible binding agents, and/or adjuvant materialscan be included as part of the composition. The tablets, pills,capsules, troches and the like can contain any of the followingingredients, or compounds of a similar nature: a binder such asmicrocrystalline cellulose, gum tragacanth or gelatin; an excipient suchas starch or lactose, a disintegrating agent such as alginic acid,Primogel, or corn starch; a lubricant such as magnesium stearate orSterotes; a glidant such as colloidal silicon dioxide; a sweeteningagent such as sucrose or saccharin; or a flavoring agent such aspeppermint, methyl salicylate, or orange flavoring.

For administration by inhalation, the compounds can be delivered in theform of an aerosol spray from a pressured container or dispenser thatcontains a suitable propellant, e.g., a gas such as carbon dioxide, or anebulizer. Such methods include those described in U.S. Pat. No.6,468,798.

Systemic administration of a therapeutic compound as described hereincan also be by transmucosal or transdermal means. For transmucosal ortransdermal administration, penetrants appropriate to the barrier to bepermeated are used in the formulation. Such penetrants are generallyknown in the art, and include, for example, for transmucosaladministration, detergents, bile salts, and fusidic acid derivatives.Transmucosal administration can be accomplished through the use of nasalsprays or suppositories. For transdermal administration, the activecompounds are formulated into ointments, salves, gels, or creams asgenerally known in the art.

The pharmaceutical compositions can also be prepared in the form ofsuppositories (e.g., with conventional suppository bases such as cocoabutter and other glycerides) or retention enemas for rectal delivery; ina crème or solid form for vaginal delivery; or in liquid form for use asa douche.

In one embodiment, the therapeutic compounds are prepared with carriersthat will protect the therapeutic compounds against rapid eliminationfrom the body, such as a controlled release formulation, includingimplants and microencapsulated delivery systems. Biodegradable,biocompatible polymers can be used, such as ethylene vinyl acetate,polyanhydrides, polyglycolic acid, collagen, polyorthoesters, andpolylactic acid. Such formulations can be prepared using standardtechniques, or obtained commercially, e.g., from Alza Corporation andNova Pharmaceuticals, Inc. Liposomal suspensions (including liposomestargeted to selected cells with monoclonal antibodies to cellularantigens) can also be used as pharmaceutically acceptable carriers.These can be prepared according to methods known to those skilled in theart, for example, as described in U.S. Pat. No. 4,522,811.

The pharmaceutical compositions can be included in a container, pack, ordispenser together with instructions for administration.

Combination Treatments

The methods described herein can also include the administration of asaponin compound described herein in combination with a therapeutic orsub-therapeutic dose of another antimycotic, e.g., clotrimazole,nystatin, fluconazole, and ketoconazole. In severe infections (e.g., inhospitalized patients), amphotericin B, caspofungin, Gentian violet, orvoriconazole may be used in combination with the compounds describedherein.

EXAMPLES

The invention is further described in the following examples, which donot limit the scope of the invention described in the claims.

Example 1 Identification of Antifungal Compounds

The ability of pathogenic fungi to overcome antifungal agents inclinical use has created a need to develop new antifungal compounds. Tofacilitate drug discovery and overcome drug development hurdles, such astoxicity and solubility, a high-throughput whole animal assay for theidentification of compounds with antifungal efficacy has been developedusing the nematode C. elegans as a heterologous host (Breger et al.,PLoS Pathog, 3:e18 (2007); Okoli et al., PLoS ONE, 4:e7025 (2009)).

The procedure for the co-inoculation antifungal compound screen wereconducted as previously described (Okoli et al., PLoS ONE, 4:e7025(2009); Tampakakis et al., Nat Protocols, 3:1925-1931 (2008)), using theC. albicans strain DAY185 (Davis et al., Infect Immun, 68:5953-5959(2000)) and the C. elegans glp-4; sek-1 double mutant.

The determination of the lowest concentration of the selected compoundsshowing in vitro antifungal activity was accomplished by following thesteps detailed in the co-inoculation assay, using two-fold serialdilutions of the test compounds. The wells were assessed by visuallymonitoring the turbidity for concentrations exhibiting in vitroinhibition of C. albicans growth.

This assay allows simultaneous assessment of a compound's potentialtoxicity and the ability to promote the survival of the nematode in thepresence of C. albicans, including modes of action not traditionallyconsidered in antifungal assays such as impeding a fungal virulencefactor or promoting host immune response. We performed a screen of 2,560natural products representing a fraction of the Analyiticon Discoverycompound collection (ac-discovery.com) housed at the Broad Institute ofHarvard and MIT (Cambridge, Mass.). Through this screen we found thatmost of our hits, defined as conferring survival to at least 20% of thenematodes after five days, were from the saponin family of naturalcompounds. These natural products identified in the primary screen wereretested and confirmed (FIG. 1; Table 1). Of the twelve saponinsidentified, six of the compounds (A7, A8, A24, A20, A17, and A21) had noprecedent in the literature regarding their structure or biologicalactivity, however in some cases related analogs have been described.Moreover, although the antifungal effects of some of these saponins havebeen reported (Sata et al., Biosci Biotechnol Biochem, 62:1904-1911(1998); Ohtani et al., Phytochem, 33:83-86 (1993)), their efficacyagainst Candida spp. has not been studied.

TABLE 1 Minimal inhibitory concentrations (MIC) in vitro and effectiveconcentration (EC₅₀) in vivo of saponins identified in the C. elegans-C.albicans screen.* Saponin natural MIC in vitro EC₅₀ in vivo products(μg/mL) (μg/mL) Amphotericin B 1.0 2.0 A2 Sakurasosaponin 27.5 55.1 A85.8 23.1 A16 Aginoside 47.0 47.0 A24 13.3 13.3 A11 38.9 38.9 A20 4.8 4.8A7 3.1 3.1 A19 26.5 26.5 A25 28.7 28.7 A17 31.0 31.0 A21 16.5 16.5*Compound A6 (Arvensoside D) was unable to provide a MIC or EC₅₀ due tothe limited antifungal activity of the compound.

After confirmation of these hits, dose response experiments wereconducted to determine the concentration that provided maximum nematodesurvival. The compounds conferred a range of nematode survival from 27%(compound A6) to 93% (compounds A2, A7, A24, and A25), however, with theexception of A6, all compounds conferred a nematode survival over 65%(FIG. 1).

Several representative members of the saponin family of natural products(compounds A8, A11, A16, A20, and A24; FIG. 1) had similar chemicalstructures and conferred a high degree of worm survival (FIG. 1).Compounds A8 and A24 are closely related analogs to the known antifungalnatural product aginoside (A16) (Sata et al., Biosci Biotechnol Biochem,62:1904-1911 (1998); Carotenuto et al., Phytochem, 51:1077-1082 (1999)).As shown in FIG. 1, A8 is the C-2 des-hydroxy analog of aginoside, andthe similar activities of A8 and A16 suggests that the C-2 oxygenationstate does not affect the overall antifungal activity. Thepentasaccacharide A24 differs from aginoside through the addition of theβ-D-glucopyranose sidechain, and this additional glycoside did confer anincrease in protection from 67-93% (FIG. 1). Similarly, compounds A11and A20, which are both characterized by oxygenation state differencesat the C-6 position in the aglycone backbone as well as differences inthe appended sugar moieties when compared to A8, A16, and A24, alsoconferred protection to the worms (FIG. 1). Clearly, a range ofglycoside substitutions is tolerated in this class of compounds andthese differences do not appear to drive the overall activity. Notably,although members of the aginoside family of saponins are well documentedin literature, there was no description of the two new glycosylatedderivatives of aginoside, A8 and A24, or a report of their antifungalactivity. It should be noted that there were several other structurallyrelated analogs composed primarily of the aglycone backbone that werenegative in the screen. Whether this is due to specific differences intheir fungicidal activity or simply a reflection of their differentphysicochemical properties (e.g. solubility) is uncertain.

Six polyglycosylated saponins were identified in the screen (A2, A7,A17, A19, A21, and A25) that completely inhibited in vitro growth of C.albicans and provided excellent protection to the worms (FIG. 1, Table1). Interestingly, several of these saponins were able to confer a levelof protection similar to that provided by amphotericin B (93%, Table 1)(Okoli et al., PLoS ONE, 4:e7025 (2009)), the current clinicalantifungal agent of choice for systemic candidasis. Of particularinterest, compound A7 was able to extend C. albicans-infected nematodesurvival to a similar level as amphotericin B, however only half of theconcentration of A7 was required (FIG. 2, panel a). There were noprevious reports in the literature describing the structure of compoundA7. Unfortunately, at high concentrations it appears that compound A7 istoxic to the nematode (FIG. 2, panel a), although there does appear tobe a therapeutic window, and modification of the compound might reduceits toxicity. Compound A2 is the known natural product sakurasosaponin,and its antifungal properties have been previously reported by Ohtari etal. (Ohtani et al., Phytochem, 33:83-86 (1993)). Compounds A17 and A21,which share a similar aglycone, are related to the maesabalide family ofcompounds; however, there were no reports for these uniquepentasaccarides that incorporate the distal furanose residue or reportsof their antifungal activity (Germonprez et al., J Med Chem, 48:32-37(2005)). Compounds A19 and A25 also demonstrated excellent in vitroactivity completely inhibiting C. albicans growth and providingexcellent nematode protection (FIG. 1; FIG. 2, panel b; Table 1). Thesesaponins share a similar aglycone core which is related to thebarrigenol family of natural products, however, there are scant reportsfor compounds displaying this arrangement of polyglycosylation (Herlt etal., J Nat Prod, 65:115-120 (2002)). There is one report in theliterature for compounds closely related to A19 (Liu et al., ChineseChem Lett, 17: 211-214 (2006)) and no references were found for compoundA25. While similar compounds have reported insect antifeedant properties(Herlt et al., J Nat Prod, 65:115-120 (2002)), there was no descriptionof their antifungal properties and, based on the potent inhibition andexcellent protective effects, we feel this class of compounds may offerunique opportunities to discover novel compounds with improved activityor inhibit novel fungal biological pathways. Collectively, therelatively “soft” structure activity relationship (SAR) demonstrated bymost the saponins is encouraging, as these compounds retained excellentantifungal potency even though there are a variety of aglyconcs,glycosides, and glycosidic linkages displayed between them.

The dose response experiments also allow the estimation of the in vitroefficacy of these compounds against C. albicans and the effective dosethat resulted in 50% survival of the nematodes (EC₅₀) was determined forthese 12 natural products (Table 1). Comparison of the concentrations ofboth the minimal inhibitory concentration (MIC) and EC₅₀ can provideinsight into possible actions the compound may have on the fungus.Compounds with a lower or equal EC₅₀ when compared to the MIC suggestthe compounds have higher efficacy during the infection process. Thiscould result from several factors including 1) immuno-modulatory effectsfrom the compounds, 2) inhibition of virulence factors, or 3) desirablesolubility and/or permeability properties of the saponins resulting inthe compounds reaching the target site effectively. Of note is thatprevious studies showed the nematode EC₅₀ concentrations of knownantifungal compounds are higher than the concentrations needed for invitro efficacy (for example the MIC for several azoles in clinical useand amphotericin B were half the concentration required to confer 50%nematode survival (Table 1; (Okoli et al., PLoS ONE, 4:c7025 (2009)).However, the saponins had identical EC₅₀ and MIC concentrations, withthe exception of compounds A2 and A8 (Table 1), suggesting their in vivoantifungal activity may also be derived by alternative mechanisms.

One explanation for the similarity in the EC₅₀ and MIC concentrations isthat the saponins may possibly alter the nematode immune response.Previous studies have demonstrated that saponins have a stimulatoryaffect on the Th1 immune response and production of cytotoxicT-lymphocytes, which has lead to their use as adjuvants in vaccines (Sunet al., Vaccine, 27:1787-1796 (2009)). It is unclear how saponins alterthis immune response, although a correlation between the length of thesugar side chain and the increase in immune stimulating ability has beenobserved, where the longer the sugar moiety, the greater IgG antibodyresponse (Sun et al., Vaccine, 27:1787-1796 (2009)). It should be notedthat eleven of the twelve saponins identified in the screen have atleast three sugars attached (FIG. 1). Although C. elegans does not havean adaptive immune response and it is currently unclear if the immuneresponse of the nematode is altered in the presence of these compounds,other studies have shown saponins induce innate immune responses;production of cytokines, such as interleukins and interferons, isincreased by saponins which may lead to stimulation of the immune system(Francis et al., British Journal of Nutrition, 88:587-605 (2002)).

Example 2 Further Characterization of Two Identified Natural Products

Two of these identified natural products (one from each group), A16(aginoside) and A19, were selected for further studies based on thefollowing considerations: (1) none of the concentrations used in thedose response experiment showed signs of toxicity to the worms (FIG. 2,panel b); (2) the compounds showed a high percentage of protection tothe worms (67% and 80% respectively) and related structural analogs fromeach class conferred the highest protection observed, 93% (A24 and A25)(FIGS. 1); and (3) the compounds were readily available from the vendor(Analyticon Discovery, Germany). Dose response experiments for A16 andA19 demonstrated dose-dependent nematode survival to C. albicansinfection up to the maximum concentration tested for the compounds (94μg/mL for A16 and 106 μg/mL for A19; FIG. 2, panel b). Using thestandard Clinical and Laboratory Standards Institute (CLSI) procedure,the in vitro MIC of these two compounds was determined on the followingC. albicans strains: DAY185 (the standard strain used throughout thescreen), two fluconazole-resistant strains of C. albicans, and anechinocandin-resistant strain of C. albicans (Table 2). Compounds A16and A19 had identical MIC values for the C. albicans isolates tested,regardless of resistance mechanisms to clinically used antifungalagents. These findings indicate that the molecular mechanisms of C.albicans which confer resistance to antifungal agents in currentclinical use do not provide cross-resistance to the natural productsidentified in this screen in agreement with other studies (Zhang et al.,Biol Pharm Bull, 28:2211-2215 (2005)). Importantly, the natural productsare likely to have a different mode of action than members of thetriazole and echinocandin family, and may be effective in treatment forisolates resistant to conventional antifungal compounds.

TABLE 2 The MIC results of clinically relevant compounds and twoidentified natural products on C. albicans.* Fluco- Ampho- Strainsnazole Caspofungin tericin B A16 A19 C. albicans strains DAY185 2 2 2 1632 Fluconazole-resistant strains 98-145 >128 1 2 16 32 95-120 32 1 2 1632 Echinocandin-resistant strain A15 2 8 2 16 16 *MIC concentrations arepresented in μg/mL

Because of the significance of biofilm in human disease (for example,biofilm formation on medical devices is associated with increasedresistance to antifungal agents (Blankenship et al., Curr OpinMicrobiol, 9:588-594 (2006); d'Enfert, Curr Drug Targets, 7: 465-470(2006); Kumamoto, Curr Opin Microbiol, 5:608-611 (2002)) we studied theeffects of saponins on Candida biofilms.

The minimal inhibitory concentration (MTC) was determined for strainsDAY185, 98-145, 95-120(White et al., Antimicrob Agents Chemother,46:1704-1713 (2002)), and A15-10 (Garcia-Effron et al., AntimicrobAgents Chemother, 53:112-122 (2009)) spectrophotometricially using RPMI1640 media (Mediatech, Inc.) following the standard CLSI microdilutionprotocol M27-A (National Committee for Clinical Laboratory Standards,Reference method for broth dilution susceptibility testing of yeasts.Tentative standard M27-A, Villanova, Pa. (1995)). Biofilm assays usingidentified compounds were conducted as previously described (Richard etal., Eukaryot Cell, 4:1493-1502 (2005)). The biofilm dry mass wasdetermined by drying the silicone squares in a chemical hood, andweighing the resulting biofilm mass subtracting the previously weighedmass of the silicone square. Biofilm pictures were captured using aconfocal laser microscope (TCS NT, Leica Microsystems). Cells were grownat 30° C., exposed to PS for 30 min, and then washed with PBS. Cellswere observed for PS localization by confocal laser microscopy (TCS; NTLeica) as described previously (Fuchs et al., Antimicrob AgentsChemother, 51:2929-2936 (2007)).

Caspofungin (Merck) served as a known antifungal compound control. Invitro hyphal inhibition was assessed by incubation of DAY185 in RPMI1640 media at 37° C. After 48 hours the cultures were visually inspectedfor hyphal formation by microscopy. The ability of the antifungalcompound A16, at either 2 or 4 μg/mL, to induce osmotic stress wasassessed using DAY185 grown in a 96 well microtiter plate containingRPMI 1640 media and NaCl, ranging in concentrations from 0-2 M in 0.25 Mincrements. The growth of the fungus was measuredspectrophotometricially after 48 hours of growth at 35° C.

Both A16 and A19 were able to inhibit biofilm formation atconcentrations below the MIC (10 and 20 μg/mL for A16 and A19,respectively) to a level comparable with the echinocandin caspofungin(FIG. 3). With the exception of the echinocandidns, most currently usedantifungal agents are unable to inhibit biofilm formation to asignificant degree.

C. albicans biofilms are composed of hyphae, pseudohyphae, yeast cells,and an extracellular matrix, where the hyphae play an integral rolewithin this complex. In order to address the reduction in biofilmformation in the presence of saponins, we tested the ability of compoundA16 to inhibit hyphae formation at various concentrations in RPMI.Untreated C. albicans is able to form extensive hyphal networks, howeverwhen C. albicans is incubated with A16 at 2 μg/mL there are very fewhyphae formed and are much smaller in size (˜5-7 cells in length). Whentreated with 1 μg/mL of A16 there is a visible reduction in the numberof hyphae, and the culture primarily consists of pseudohyphae and yeastcells.

Example 3 Hemolysis Studies

Representatives of the saponins family are able to disrupt cellularmembranes and the lytic activity on erythrocytes has been used as anassay for some saponins Francis et al., British Journal of Nutrition,88:587-605 (2002)). This property is derived from the affinity of somesaponins for binding cholesterol forming insoluble pores composed of thesterol and saponins (Bangham et al., Nature, 196:952-953(1962); Glauertet al., Nature, 196:953-955 (1962)). Although the hemolytic propertiesof saponins have been well documented, several saponins are now known tohave little or no hemolytic activity (Sun et al., Vaccine, 27:1787-1796(2009); Francis et al., British Journal of Nutrition, 88:587-605(2002)). The dose-response experiments previously used to determine theEC₅₀ and approximate the MIC can also indicate if the saponins maybetoxic to C. elegans and potentially to mammalian cells. The compound maypotentially be toxic to the nematode if a decrease in C. eleganssurvival is observed despite an increase in the concentration of thecompound.

The cytotoxicity for the identified compounds was confirmed aspreviously described (Breger et al., PLoS Pathog, 3:e18 (2007); Moy etal., Proc Natl Acad Sci USA, 103:10414-10419 (2006)). Hemolysis of sheeperythrocytes (Rockland Immunochemicals) was monitored on aspectrophotometer at A₅₄₀ with the two natural products A16 and A19 (100μg/mL) in 2% DMSO. Triton X-100 and DMSO were used as controls.

Of the 12 saponins conferring an increase in C. elegans survival, onlyA7 and A24 displayed a decrease in nematode survival when tested athigher concentrations (FIG. 2, panel a; data not shown). This trendsuggests the saponins could be toxic at high concentrations, althoughboth were able to confer 93% nematode survival at a lower concentration.The in vivo nature of this antifungal discovery assay may have limitedthe number of toxic saponins identified in the screen, as they may havebeen toxic to the nematode during the screening process. Importantly,hemolysis experiments using sheep erythrocytes and the two purchasedsaponins (A16 and A19) demonstrated no hemolytic activity at 100 μg/mL,a concentration which is at least three times the MIC for C. albicansDAY185.

The aglycone backbone of saponins is believed to play a role inhemolysis as this core has an affinity for cholesterol (Glauert et al.,Nature, 196:953-955 (1962)). The saponin aglycone structure can bedivided into the triterpenoid and steroidal structural subclasses (Sparget al., J Ethnopharmacol, 94:219-243(2004)), where steroidal saponinshave higher hemolytic activity and hemolysis occurs at a faster ratewhen compared to triterpenoid saponins (Takechi et al., Planta Med,61:76-77 (1995)). All 12 compounds identified in the assay weretriterpenoid based saponins and may explain why only two compoundsdisplayed potential toxicity in C. elegans. Other studies have suggestedthe hemolytic properties of saponins could be due to several factorsincluding the types of side chains and the number of appended glycosidesand polar functional groups present in the aglycone (Francis et al.,British Journal of Nutrition, 88:587-605 (2002)). Compound A24 was theonly compound in this group that showed evidence of toxicity to theworms at concentrations >27 μg/mL, suggesting that while the antifungalactivity is relatively conserved with a range of glycoside substitutionpatterns, toxicity may be related to the differences in pendant sugarmoieties rather than the core triterpenoid aglycone.

Example 4 Osmotic Stress and Potentiation of Photodynamic Inactivationin C. albicans by A16

Some saponins are capable of forming pores in Saccharomyces cerevisiaemembranes by binding to the fungal sterol ergosterol causing cellularleakage (Simons et al., Antimicrob Agents Chemother, 50:2732-2740(2006)). To investigate if these saponins increase cellular leakage andpermeability, the potential of compound A16 to increase thesusceptibility of the fungus to osmotic stress and enhance photodynamicinactivation (PDI) was assessed.

The PS used were Rose Bengal (RB, Sigma-Aldrich, St. Louis, Mo.) andchlorin(e6) (ce6, Frontier Scientific, Logan, Utah). PEI-ce6 wassynthesized as a covalent conjugate between polyethylenimine (MW range10,000-25,000, an average of one ce6 per chain) and ce6 as describedpreviously (Tegos et al., Antimicrob Agents Chemother, 50:1402-1410(2006)). Stock solutions were prepared in water at a concentration of 2mM and stored for a maximum of 2 weeks in the dark at 4° C. before use.Spectra of stock solutions of PS diluted 140- to 280-fold in methanolwere recorded. A noncoherent light source with interchangeable fiberbundles (LC122; LumaCare, London, United Kingdom) was employed.Thirty-nanometer-band-pass filters at ranges of 540±15 nm for RB, and660±15 nm for ce6 and PEI-ce6 were used. The total power output from thefiber bundle ranged from 300 to 600 mW. The spot was arranged to give anirradiance of 100 mW/cm².

The statistical values for the PDI experiments represent the mean ofthree separate experiments, and bars presented in the graphs representstandard error from the mean. Differences between mean values weretested for significance by an unpaired two-tailed Student t test,assuming equal or unequal variations as appropriate. A P value of lessthan 0.05 indicated statistical significance.

The C. albicans cell wall and membrane are important for osmoregulationto maintain proper physiological conditions to carryout enzymaticreactions. Since saponins are able to disrupt the fungal cell membrane,external osmotic stress should also have detrimental effects on thefungal cell. C. albicans was grown in the presence of 2 μg/mL ofcompound A16 under various salt concentrations to assess the effect thesaponin has on salt induced osmotic stress.

Fungal suspensions in phosphate-buffered saline (PBS) (initialconcentration, 10⁸ CFU ml⁻¹) were pre-incubated with A16 for 1 and 24hrs in combination with the appropriate PS in the dark at roomtemperature for 30 min at concentrations varying from 10 to 100 μM forthe PS and 4 μg/ml for A16. The cell suspensions were centrifuged at12,000 rpm, washed twice, and suspended in sterile PBS. The cellsuspensions were placed in wells of 48-well microtiter plates (FisherScientific) and illuminated using appropriate optical parameters.Fluences ranged from 0 to 80 J/cm² at a fluence rate of 100 mW/cm².During illumination, aliquots of 100 μL were taken to determine the CFU.The contents of the wells were constantly stirred during illumination toensure that cells did not settle to the bottom of the wells and mixedbefore sampling. The aliquots were serially diluted 10-fold in PBS andwere streaked horizontally on square YPD agar plates as described (Jettet al., Biotechniques, 23:648-650 (1997)). Plates were incubated at 30°C. for 48 hrs. Two types of control conditions were used: illuminationin the absence of PS or A16 and incubation with PS and A16 in the dark.

The fungus incubated with A16 was unable to grow at a high saltconcentration when compared to the untreated control (0.5 M for the A16treated versus 1.25 M for the untreated control) demonstrating anincreased sensitivity to NaCl induced osmotic stress.

Photodynamic inactivation utilizes a non-toxic dye, or photosensitizer(PS), which is able to generate reactive oxygen species, such as singletoxygen and hydroxyl radical, in the presence of oxygen and low-intensitylight of the correct wavelength to be absorbed by the PS ultimatelyproducing toxic effects in microbial cells (Fuchs et al., AntimicrobAgents Chemother, 51:2929-2936 (2007)). The application of PDI andphotodynamic therapy (PDT) as an antimicrobial treatment is a developingarea of photobiology and has been investigated as a highly promisingpotential treatment for localized infections (Demidova et al., Int JImmunopathol Pharmacol, 17: 245-254 (2004); Hamblin et al., PhotochemPhotobiol Sci, 3:436-450 (2004)). Three different PSs molecules wereused for studies with compound A16: two were anionic, rose bengal (RB),chlorin(e6) (ce6), while the third was a polycationic conjugate of ce6and polyethyleneimine (PEI-ce6). Both RB and ce6 are not taken up easilyby yeast cells and at the concentrations used they had no statisticallysignificant PDI effect (Fuchs et al., Antimicrob Agents Chemother,51:2929-2936 (2007); Tegos et al., Antimicrob Agents Chemother,50:196-203(2006)). However, PEI-ce6 is more potent at lowerconcentrations than the other PS compounds against C. albicans and C.neoformans (Fuchs et al., Antimicrob Agents Chemother, 51:2929-2936(2007); Tegos et al., Antimicrob Agents Chemother, 50:196-203(2006)),due to its increased ability to disrupt and pass through the cell wall.The survival fraction and uptake (molecules/cell) of C. albicans wasdetermined for PD1 mediated by the 3 different PSs with or without A16pre-incubation for various time periods ranging between 1-24 hours. C.albicans was incubated with 100 μM RB, 100 μM ce6, or 10 μM PEI-ce6 for30 min in either the presence or absence of a sub-inhibitoryconcentration (4 μg/mL) of A16 and received increasing fluences of 540nm or 660 nm of light. The light-dependent killing of C. albicans in thepresence of A16 was 2 to 5 logs greater than the killing at the samefluence in the absence of A16 for both RB and ce6 (FIG. 4, panels a,b).There was virtually no effect on the yeast cells either in the presenceor absence of light. Killing by PEI-ce6 mediated PDI does not changedramatically after pre-incubation with the compound (FIG. 4, panel c).This observation is consistent with the fact that PEI-ce6, due to itspolycationic charge, is self sufficient in bypassing the cellpermeability barrier.

In order to confirm that the increase in phototoxicity observed bycombining the different PS with A16 is actually due to an increase ofcellular uptake of the PS by the cells and to document that theantifungal efficacy of saponins against C. albicans is associated withincreased permeability, the amount of dye within the cell was measuredby fluorescence spectrofluorimetry.

Cell suspensions (10⁸ cells/mL) were incubated in the dark as aboveusing the same concentrations as for the PDI assays measured as μM PSequivalent (final concentration in incubation medium). Incubations werecarried out in triplicate. Cell suspensions were centrifuged (12000 rpm,1 min) and washed twice in 1 mL sterile PBS. The cell pellet wasdissolved in 1.5 mL 0.1 M NaOH/1% SDS for 24 h to yield a homogenoussolution. Uptake was determined by measurement of fluorescence in black96-well flat-bottom plates (Costar) in a final volume of 200 μL using aSpectramax Gemini spectrofluorimeter (Molecular Devices) at 400nm_(ex)/580-700 nm_(em) for ce6, PEI-ce6 and 552 nm_(ex)/555-620 nm_(em)for RB. Uptake values were obtained as previously described (Tegos etal., Antimicrob Agents Chemother, 50:196-203(2006)).

The cellular uptake of dye can be expressed as molecules per cell bycorrelation of the extracted PS concentration with the number of C.albicans cells present. In each case the addition of compound A16dramatically increased the uptake of PS by the cells, and thesedifferences were statistically significant (Table 3).

TABLE 3 Uptake assessment of photosensitizers in the presence of thenatural product A16. PS +A16 (1 hr) +A16 (24 hrs) RB 3.8 ± 0.6 4.55 ±0.5*  9.12 ± 0.22** ce6 0.81 ± 0.06  3.1 ± 0.25**  9.1 ± 0.15** PEI-ce613.4 ± 1.1  20.9 ± 1.3** 23.0 ± 0.7**  Values represent the uptake inmolecules/cell from pellets obtained after incubation of the cellsuspensions with different PS with or without A16 at the sameconcentrations used for PDI studies. Values are the means of threedeterminations + standard deviation. The yeast cell density was, 10⁸CFU/ml. **P < 0.01; and, *P < 0.1; compared with the uptake values of PSalone.

The influx of the PS ce6 into C. albicans cells was further examined andconfirmed by confocal laser scanning microscopy. Alone, ce6 demonstratedno apparent internalization into either yeast or pseudohyphae C.albicans cells after one hour incubation (FIG. 5, panel a). However,when ce6 and A16 are incubated together for one hour, internalization ofce6 is visible (FIG. 5, panel b). Consistent with the PDI assays, aftera 24 hour incubation period there was a dramatic increase in theconcentration of ce6 inside the fungal cells in both yeast form (FIG. 5,panel c) and, in particular, pseudohyphae (FIG. 5, panel d), furtherconfirming the ability of A16 to increase cell permeability.

The pore-forming characteristic of saponins makes them ideally suitedfor use with conventional antifungal therapy. Compound A16 was able toincrease uptake of PS enabling much increased PDI of the fungus. Here weshow that saponins in conjunction with PDT may be used for treatment ofC. albicans infections. This study is the first to demonstrate that RBand ce6 in the presence with a saponin have a dramatic PDI effect tofungal cells (FIG. 4, panels a-c; FIG. 5, panels a-d; Table 3).Furthermore, the dramatic increase in permeability of pseudohyphae (FIG.5, panel d), when compared to C. albicans cells in the yeast morphology(FIG. 5, panel c), suggests the previously observed decrease in biofilmformation in the presence of the saponin (FIG. 3) is due to an increasein permeability of pseudohyphae and hyphae.

Example 5 Interaction Between A16 and Fluconazole

Since compound A16 was able to facilitate uptake of PS compounds, weinvestigated their ability to increase uptake to the commonly usedantifungal agent fluconazole. As indicated above, fluconazole andsaponins have different target sites, although they both function byaltering the fungal cell membrane. Interestingly, when we exposed fungito different concentrations of fluconazole and A16, we found that asubinhibitory concentration of compound A16 (4 μg/mL) was able todecrease the MIC of C. albicans isolate DAY185 from 2 μg/mL forfluconazole alone to 1 μg/mL for a combination of the two compounds.Despite less growth in the well resulting in a “speckled” pattern, thetwo C. albicans isolates with increased resistance to fluconazole showedno increase in sensitivity to fluconazole treatment in the presence ofA16. One of the molecular mechanisms responsible for increasedresistance to fluconazole for isolate 98-145 is a homozygous V437I pointmutation in the ERG11 gene (White et al., Antimicrob Agents Chemother,46:1704-1713 (2002)). This suggests that the alteration of thefluconazole target site still renders the fungus resistant despite apotential increase in influx of fluconazole caused by addition ofcompound A16.

The saponins were able to inhibit growth of several C. albicansisolates, including isolates which were resistant to fluconazole andechinocandins (Table 2). Whether or not C. albicans can developresistance to saponins is not known. Since saponins are synthesizedmostly by plants, plant pathogenic fungi have developed resistancemechanisms to these natural products (Osbourn, Trends Plant Sci, 1:4-9(1996)). There are several mechanisms in which phytopathogenic fungievade saponin toxicity, ranging from avoidance to enzymatic degradation(Osbourn, Trends Plant Sci, 1:4-9 (1996)). Studies using S. cerevisiaeand the saponin α-tomatine from tomato have shown that the fungus hasgreater inhibition to a degradation product, tomatidine, than to thecomplete α-tomatine saponin (Simons et al., Antimicrob Agents Chemother,50:2732-2740 (2006)), suggesting that if C. albicans gains/evolves theability to detoxify saponins, it may still be inhibited by thedegradation products.

The abundance of saponin derived natural products and the lack of overtcellular toxicity displayed by the majority of compounds in this studysuggests saponins may provide a promising source of new antifungalagents. These compounds represent an opportunity to expand the currentclasses of antifungal agents in use and to improve available antifungaldrugs by exploiting these new chemical scaffolds. Future studies willfocus on defining the minimal structural components required to retainfull inhibitory and protective effects against C. albicans.

Other Embodiments

It is to be understood that while the invention has been described inconjunction with the detailed description thereof, the foregoingdescription is intended to illustrate and not limit the scope of theinvention, which is defined by the scope of the appended claims. Otheraspects, advantages, and modifications are within the scope of thefollowing claims.

1-6. (canceled)
 7. A method of treating a fungal infection in a subject,the method comprising administering to the subject a therapeuticallyeffective amount of a compound of Formula II:

or a pharmaceutically acceptable salt thereof, wherein: R₁ is H orOC(O)R^(A); R₂ is H or OH; R₃ is H, C(O)OR^(A), CH₂OR^(A),CH₂OC(O)R^(A); R₄ and R₅ are each independently H or OC(O)R^(B); R^(A)is H or C₁₋₆ alkyl; R^(B) is C₂₋₆ alkenyl; and Gly is


8. The method of claim 7, wherein the compound is:

or a pharmaceutically acceptable salt thereof.
 9. The method of claim 7,wherein the compound is:

or a pharmaceutically acceptable salt thereof.
 10. The method of claim7, wherein the compound is:

or a pharmaceutically acceptable salt thereof.
 11. The method of claim7, wherein the compound is:

or a pharmaceutically acceptable salt thereof. 12-18. (canceled)
 19. Themethod of claim 7, wherein the fungal infection is infection with aCandida species fungus.